Carbohydrate-lipid constructs and their use in preventing or treating viral infection

ABSTRACT

The invention relates to selected carbohydrate-lipid constructs and their use as mimics of ligands for receptors expressed by virus. In particular, the invention relates to the use of selected carbohydrate-lipid constructs in methods of inhibiting virus infection and/or promoting clearance of virus from infected subjects. Carbohydrate-lipid constructs selected for use in these methods where the virus is Human Immunodeficiency Virus (HIV) are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 13/459,399filed Apr. 30, 2012, which is a continuation of application Ser. No.12/451,120 filed Mar. 29, 2010 (U.S. Pat. No. 8,211,860) which is a 371of PCT/NZ2008/000095 filed Apr. 28, 2008 which claims priority to NewZealand Application Nos. 554853 filed Apr. 27, 2007; 556736 filed Jul.24, 2007, and 567754 filed Apr. 24, 2008, the entire contents of each ofwhich are hereby incorporated by reference.

TECHNICAL FIELD

The invention relates to selected carbohydrate-lipid constructs andtheir use as mimics of ligands for receptors expressed by a virus.

In particular, the invention relates to the use of selectedcarbohydrate-lipid constructs in methods of inhibiting viral infectionand/or promoting clearance of virus from infected subjects.

Carbohydrate-lipid constructs selected for use in these methods wherethe virus is Human Immunodeficiency Virus (HIV) are provided.

BACKGROUND ART

Infection with HIV and Acquired Immune Deficiency Syndrome (AIDS)continues to increase worldwide, despite intense research to control itsspread. Furthermore, the emergence of new viral infections presentsadditional challenges to public health.

Therapies to treat infection that target viruses may be limited inefficacy due to resistance and genetic variance of the virus.

HIV infection is mediated by the viral fusion glycoprotein gp120-gp41binding the cell surface expressed receptor CD4. This binding is thebasis of the viral targeting of T lymphocytes and monocyte macrophages.The receptor gp120 shows an affinity in vitro for severalglycosphingolipids (GSLs) (Ghat et al (1993); Fantini et al (1998);Mylvaganam and Lingwood (1999a)).

A need exists for glycolipid mimics that are dispersible inbiocompatible media and can be used to modify the interaction betweennaturally occurring membrane incorporated glycoconjugates, such as GSLs,and the receptors expressed by a virus. Such water soluble glycolipidmimics have been recognized as having potential for use in thepreventative treatment of individuals at risk of infection from virusessuch as HIV.

Lund et al (2006) have investigated the effect of the water solubleglycolipid mimic adamantylGb₃ on HIV infection of cells in culture. Inprevious studies adamantylGb₃ had been demonstrated to be a superiorligand for the receptor gp120 (Mahfoud et al (2002)).

A dose dependent inhibition of infection of Jurkat T cells by HIV-1pre-incubated with adamantylGb₃ has been demonstrated in vitro (Lund etal 2006). The in vivo inhibition of infection by HIV-1 was not reported,but the water soluble glycolipid mimic was indicated to have no effecton Jurkat T cell viability. Transient changes in CD4 surface expressionwere observed. Lund et al (2006) attributed the dose dependentinhibition of infection to an inhibition of attachment of thepre-treated HIV-1 to the Jurkat T cells. The adamantylGb₃ treated virusremained non-host cell attached and virions could not be found withinthe Jurkat cells.

Further studies on HIV-1 infection of primary lymphoid cells in vitroprovided results consistent with those observed for Jurkat T cells ashost cells. Infection by both wild type and drug resistant HIV-1infection was inhibited by the pre-treatment of the water solubleglycolipid mimic adamantylGb₃. However, pre-incubation of cells withadamantylGb₃ was ineffective.

Lund et al (2006) noted the effective concentration range required toinhibit HIV-1 infection would be difficult to maintain clinically, butsuggested the formulation of adamantylGb₃ within a cream might provide atopical ointment for the prevention of mucosal HIV infection.

It is an object of the invention to provide receptor bindingcarbohydrate-lipid constructs that are effective to inhibit viralinfection of the cells of a subject.

It is a further object of the invention to provide receptor bindingcarbohydrate-lipid constructs that are effective to promote clearance ofvirus from an infected subject.

These objects are to be read disjunctively with the object of to atleast provide a useful choice.

DISCLOSURE OF INVENTION

In a first aspect the invention provides a method of inhibitinginfection of the cells of a subject by a virus by administering to thesubject an amount of carbohydrate-lipid construct of the formulaF-S₁-S₂-L where:

-   -   F is selected from the group consisting of glycotopes of ligands        for one or more receptors expressed by the virus;    -   S₁-S₂ is a spacer linking F to L; and    -   L is a lipid selected from the group consisting of diacyl- and        dialkyl-glycerolipids, including glycerophospholipids.

Preferably, the amount is effective to inhibit binding of the receptorexpressed by the virus to a cell surface expressed ligand.

Preferably, the receptor is expressed by the human immunodeficiencyvirus (HIV).

S₁-S₂ is selected to provide a carbohydrate-lipid construct that isdispersible in water.

Preferably, S₁ is a C₂₋₅-aminoalkyl selected from the group consistingof: 2-aminoethyl; 3-aminopropyl; 4-aminobutyl; and 5-aminopentyl.

Preferably, S₂ is selected from the group consisting of: —CO(CH₂)₃CO—;—CO(CH₂)₄CO-(adipate); and —CO(CH₂)₅CO—.

Preferably, L is selected from the group consisting of:diacylglycerolipids, phosphatidate, phosphatidyl choline, phosphatidylethanolamine, phosphatidyl serine, phosphatidyl inositol, phosphatidylglycerol, and diphosphatidyl glycerol derived from one or more oftrans-3-hexadecenoic acid, cis-5-hexadecenoic acid, cis-7-hexadecenoicacid, cis-9-hexadecenoic acid, cis-6-octadecenoic acid,cis-9-octadecenoic acid, trans-9-octadecenoic acid,trans-11-octadecenoic acid, cis-11-octadecenoic acid, cis-11-eicosenoicacid or cis-13-docosenoic acid. More preferably, the lipid is derivedfrom one or more cis-desaturated fatty acids. Most preferably, L isselected from the group consisting of:1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) and1,2-O-distearyl-sn-glycero-3-phosphatidylethanolamine (DSPE); andrac-1,2-dioleoylglycerol (DOG).

Preferably, L is a glycerophospholipid and the construct includes thesubstructure:

where X is H or C, * is other than H and n is an integer 2 to 5.

More preferably, L is a glycerophospholipid and the construct includesthe substructure:

where:

-   -   X is H;    -   R₁ is a C_(p)-alkyl glycoside,    -   R₂ and R₃ are independently selected from the group consisting        of: trans-3-hexadecenal, cis-5-hexadecenal, cis-7-hexadecenal,        cis-9-hexadecenal, cis-6-octadecenal, cis-9-octadecenal,        trans-9-octadecenal, trans-11-octadecenal, cis-11-octadecenal,        cis-11-eicosenal and cis-13-docsenal;    -   n is 2 to 5; and    -   p is 2 or 3.

Most preferably, the glycoside is1-O—(O-α-D-galactopyranosyl-(1→4)-O-β-D-galactopyranosyl-(1→4)-β-D-glucopyranosyl(Gb₃), n is 4 and p is 3.

In specific embodiments of the first aspect of the invention thecarbohydrate-lipid construct has the structure:

designated Gb₃-sp3-Ad-DOPE (I); the structure:

designated Gb₃-sp3-Ad-DSPE (II); the structure:

designated Gb₃-sp2-Ad-DOPE (III); or the structure:

designated Gb₃-sp2-Ad-DSPE (IV).

In a first embodiment of the first aspect of the invention theadministering to the subject is by intravascular injection. Preferably,the administering is by intravenous injection.

Preferably, the administering to the subject is to provide aconcentration in the plasma of the subject of greater than 400 μM.

In a second embodiment of the first aspect of the invention theadministering to the subject is by topical application. Preferably, theadministering to the subject is by topical application as a cream orsuppository.

In a second aspect the invention provides a method of promotingclearance of a virus from an infected subject by administering to thesubject an amount of carbohydrate-lipid construct of the formulaF-S₁-S₂-L where:

-   -   F is selected from the group consisting of glycotopes of ligands        for one or more receptors expressed by the virus;    -   S₁-S₂ is a spacer linking F to L; and    -   L is a lipid selected from the group consisting of diacyl- and        dialkyl-glycerolipids, including glycerophospholipids.

Preferably, the administering to the subject is by intravascularinjection. More preferably, the administering is by intravenousinjection.

Preferably, the amount is sufficient to promote partitioning of thecarbohydrate-lipid construct into the membranes of cells of the vascularsystem.

Preferably, the administering to the subject is to provide an initialconcentration in the plasma of the subject of greater than 400 μM.

Preferably, the receptor is expressed by the human immunodeficiencyvirus (HIV).

S₁-S₂ is selected to provide a carbohydrate-lipid construct that isdispersible in water.

Preferably, S₁-S₂ is selected to provide a carbohydrate-lipid constructthat partitions into a lipid bi-layer when a solution of the constructis contacted with the lipid bi-layer.

Preferably, S₁ is a C₂₋₅-aminoalkyl selected from the group consistingof: 2-aminoethyl, 3-aminopropyl, 4-aminobutyl, or 5-aminopentyl.

Preferably, S₂ is selected from the group consisting of: —CO(CH₂)₃CO—,—CO(CH₂)₄CO-(adipate), —CO(CH₂)₅CO— and —CO(CH₂)₅NHCO(CH₂)₅CO—.

Preferably, L is selected from the group consisting of:diacylglycerolipids, phosphatidate, phosphatidyl choline, phosphatidylethanolamine, phosphatidyl serine, phosphatidyl inositol, phosphatidylglycerol, and diphosphatidyl glycerol derived from one or more oftrans-3-hexadecenoic acid, cis-5-hexadecenoic acid, cis-7-hexadecenoicacid, cis-9-hexadecenoic acid, cis-6-octadecenoic acid,cis-9-octadecenoic acid, trans-9-octadecenoic acid,trans-11-octadecenoic acid, cis-11-octadecenoic acid, cis-11-eicosenoicacid or cis-13-docsenoic acid. More preferably, the lipid is derivedfrom one or more cis-desaturated fatty acids. Most preferably, L isselected from the group consisting of:1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) and1,2-O-distearyl-sn-glycero-3-phosphatidylethanolamine (DSPE); andrac-1,2-dioleoylglycerol (DOG).

Preferably, L is a glycerophospholipid and the construct includes thesubstructure:

where X is H or C, * is other than H and n is an integer 2 to 5.

More preferably, L is a glycerophospholipid and the construct includesthe substructure:

where:

-   -   X is H;    -   R₁ is a C_(p)-alkyl glycoside,    -   R₂ and R₃ are independently selected from the group consisting        of: trans-3-hexadecenal, cis-5-hexadecenal, cis-7-hexadecenal,        cis-9-hexadecenal, cis-6-octadecenal, cis-9-octadecenal,        trans-9-octadecenal, trans-11-octadecenal, cis-11-octadecenal,        cis-11-eicosenal or cis-13-docsenal;    -   n is 2 to 5; and    -   p is 2 or 3.

Most preferably, the glycoside is1-O—(O-α-D-galactopyranosyl-(1→4)-O-β-D-galactopyranosyl-(1→4)-β-D-glucopyranosyl(Gb₃), n is 4 and p is 3.

In specific embodiments of the second aspect of the invention thecarbohydrate-lipid construct has the structure:

designated Gb₃-sp3-Ad-DOPE (I); the structure:

designated Gb₃-sp3-Ad-DSPE (II); the structure:

designated Gb₃-sp2-Ad-DOPE (III); or the structure:

designated Gb₃-sp2-Ad-DSPE (IV).

In a third aspect the invention provides a pharmaceutical preparationfor administration to a subject comprising a receptor bindingcarbohydrate-lipid construct of the formula F-S₁-S₂-L where:

-   -   F is selected from the group consisting of glycotopes of ligands        for a receptor;    -   S₁-S₂ is a spacer linking F to L; and    -   L is a lipid selected from the group consisting of diacyl- and        dialkyl-glycerolipids, including glycerophospholipids; and        pharmaceutically acceptable formulants.

Preferably, the receptor is expressed by a virus. More preferably, thereceptor is expressed by the human immunodeficiency virus (HIV).

Preferably, the pharmaceutical preparation is in the form of a cream orsuppository.

S₁-S₂ is selected to provide a carbohydrate-lipid construct that isdispersible in water.

Preferably, S₁-S₂ is selected to provide a carbohydrate-lipid constructthat partitions into a lipid bi-layer when a solution of the constructis contacted with the lipid bi-layer.

Preferably, S₁ is a C₂₋₅-aminoalkyl selected from the group consistingof: 2-aminoethyl, 3-aminopropyl, 4-aminobutyl, or 5-aminopentyl.

Preferably, S₂ is selected from the group consisting of: —CO(CH₂)₃CO—;—CO(CH₂)₄CO-(adipate); and —CO(CH₂)₅CO—.

Preferably, L is selected from the group consisting of:diacylglycerolipids, phosphatidate, phosphatidyl choline, phosphatidylethanolamine, phosphatidyl serine, phosphatidyl inositol, phosphatidylglycerol, and diphosphatidyl glycerol derived from one or more oftrans-3-hexadecenoic acid, cis-5-hexadecenoic acid, cis-7-hexadecenoicacid, cis-9-hexadecenoic acid, cis-6-octadecenoic acid,cis-9-octadecenoic acid, trans-9-octadecenoic acid,trans-11-octadecenoic acid, cis-11-octadecenoic acid, cis-11-eicosenoicacid or cis-13-docosenoic acid. More preferably, the lipid is derivedfrom one or more cis-desaturated fatty acids. Most preferably, L isselected from the group consisting of:1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) and1,2-O-distearyl-sn-glycero-3-phosphatidylethanolamine (DSPE); andrac-1,2-dioleoylglycerol (DOG).

Preferably, L is a glycerophospholipid and the construct includes thesubstructure:

where X is H or C, * is other than H and n is an integer 2 to 5.

More preferably, L is a glycerophospholipid and the construct includesthe substructure:

where:

-   -   X is H;    -   R₁ is a C_(p)-alkyl glycoside,    -   R₂ and R₃ are independently selected from the group consisting        of: trans-3-hexadecenal, cis-5-hexadecenal, cis-7-hexadecenal,        cis-9-hexadecenal, cis-6-octadecenal, cis-9-octadecenal,        trans-9-octadecenal, trans-11-octadecenal, cis-11-octadecenal,        cis-11-eicosenal or cis-13-docsenal;    -   n is 2 to 5; and    -   p is 2 or 3.

Most preferably, the glycoside is1-O—(O-α-D-galactopyranosyl-(1→4)-O-β-D-galactopyranosyl-(1→4)-β-D-glucopyranosyl(Gb₃), n is 4 and p is 3.

In specific embodiments of the third aspect of the invention thecarbohydrate-lipid construct has the structure:

designated Gb₃-sp3-Ad-DOPE (I); the structure:

designated Gb₃-sp3-Ad-DSPE (II); the structure:

designated Gb₃-sp2-Ad-DOPE (III); or the structure:

designated Gb₃-sp2-Ad-DSPE (IV).

In a first embodiment of the third aspect of the invention thepharmaceutical preparation is formulated for administration byintravascular injection. Preferably, the pharmaceutical preparation isformulated for administration by intravenous injection. More preferably,the pharmaceutical preparation is formulated as an aqueous formulation.Yet more preferably, the pharmaceutical preparation is a suspension ofred blood cells of the subject modified to incorporate the receptorbinding carbohydrate-lipid construct. Most preferably, thepharmaceutical preparation is identified for use in inhibiting HIVinfection and/or promoting clearance of HIV from infected subjects.

In a third embodiment of the third aspect of the invention thepharmaceutical preparation is formulated for administration as a creamor suppository. Preferably, the pharmaceutical preparation is formulatedfor administration as a cream. More preferably, the pharmaceuticalpreparation is formulated as an aqueous formulation. Most preferably,the pharmaceutical preparation is identified for use in inhibiting orpreventing HIV infection.

In the description and claims of the specification the following termsand phrases have the meaning provided:

“Carbohydrate-lipid construct” means a synthetic molecule used as aglycolipid mimic.

“Gb3” means the carbohydrate portion of the ganglioside Gb3 (ChemicalAbstract Service (CAS) REGISTRY number 71965-57-6)

“C_(p)-alkyl glycoside” means an alkyl glycoside consisting of anunbranched chain of p methylene units attached to the carbohydrate via aglycosidic linkage as exemplified by the propyl glycoside (p is 3) ofthe structure:

designated Gb₃-sp3.

“Dispersible in water” means a stable, single phase dispersion of thecarbohydrate-lipid construct may be prepared in water at a concentrationof up to at least 1000 μM in the absence of organic solvents ordetergents.

“Glycotope” means the portion of the carbohydrate moiety of a ligandthat associates with the binding site of a receptor.

“Ligand” means any molecule or portion of a molecule that binds to oneor more macromolecules, such as surface expressed antigens.

“Pharmaceutically acceptable formulants” means ingredients included inthe formulation of a pharmaceutical composition.

“Receptor” means a macromolecule or portion of a macromolecule such as asurface expressed antigen that binds to one or more ligands.

“Vascular system” means the system of vessels that convey fluids such asblood or lymph, or provide for the circulation of such fluids.

In the context of administering to the subject to provide a specifiedconcentration in the plasma of the subject the administering may be byrepeated administration to maintain the specified concentration in theplasma.

From the structures and substructures of the carbohydrate-lipidconstructs it will be recognised that M is typically H, but may bereplaced by another monovalent cation such as Na⁺, K⁺, NH₄ ⁺ andtriethylamine ([NH(CH₂CH₃)₃]⁺), and the secondary amino functions of thecarbohydrate-lipid construct may be protonated. The carbohydrate-lipidconstructs may be prepared as a range of pharmaceutically acceptablesalts.

Where the suffix “-al” is employed in respect of the substituents R₂ andR₃, an aldehyde structure is intended as exemplified bycis-9-octadecenal of the structure:

The invention will now be described in detail with reference to examplesand the Figures of the accompanying drawings pages that are indicativeof the utility of the subject matter claimed in the treatment of humansubjects.

BRIEF DESCRIPTION OF FIGURES

FIG. 1. ¹H-NMR data for the carbohydrate-lipid construct designatedGb₃-sp3-Ad-DOPE (I).

FIG. 2. Effect of 250 μM Gb₃-sp3-Ad-DOPE (I) on VSV/HIV infection inJurkat Cells (RLU): A—Control; B—AZT; C—VSV/HIV; and D—250 μMGb₃-sp3-Ad-DOPE (I).

FIG. 3. Inhibition of infection of Jurkat cells by pre-incubation of X4HIV-1_(IIIB) with the carbohydrate-lipid construct designatedGb₃-sp3-Ad-DOPE (I) (p24 pg/mL)(r=4): A—Control; B—50 μM; C—100 μM;D—200 μM; E—400 μM; F—600 μM; G—800 μM; and H—1000 μM.

FIG. 4. Inhibition of infection of Jurkat cells by pre-incubation of X4with the carbohydrate-lipid construct designated Gb₃-sp3-Ad-DOPE (I)(p24 pg/mL)(r=3): A—Control; B—50 μM; C—100 μM; D—200 μM; E—400 μM;F—600 μM; G—800 μM; and H—1000 μM.

FIG. 5. Inhibition of infection of peripheral blood mononuclear cells bypre-incubation of R5 HIV-1_(Ba-L) with the carbohydrate-lipid constructdesignated Gb₃-sp2-Ad-DOPE (III) (p24 pg/mL)(r=4).

FIG. 6. Inhibition of infection of peripheral blood mononuclear cells bypre-incubation of X4 HIV-1_(IIIB) with the carbohydrate-lipid constructdesignated Gb₃-sp2-Ad-DOPE (III) (p24 pg/mL)(r=4).

FIG. 7. Infection of Jurkat cells by pseudoenvelope-typedVSV-G/NL4-3lucHIV-1 (luciferase assay).

FIG. 8. Infection of NIH3T3 cells by pseudoenvelope-typedVSV-G/NL4-3lucHIV-1 (luciferase assay).

FIG. 9. Infection of (a) Jurkat cells and (b) NIH3T3 cells bypseudoenvelope-typed VSV-G/NL4-3lucHIV-1 (luciferase assay).

FIG. 10. Infection of (a) NIH3T3 cells and (b) Jurkat cells bypseudoenvelope-typed VSV-G/NL4-3lucHIV-1 (PCR).

FIG. 11. Inhibition of infection of rectal mucosa by VSV/HIV byapplication of a carbopol-based gel containing 3 mM thecarbohydrate-lipid construct designated Gb₃-sp2-Ad-DOPE (III)(copynumber HIV-1 cDNA)(n=4).

FIG. 12. Inhibition of infection of vaginal mucosa by VSV/HIV byapplication of a carbopol-based gel containing 3 mM thecarbohydrate-lipid construct designated Gb₃-sp2-Ad-DOPE (III)(copynumber HIV-1 cDNA)(n=4).

FIG. 13. Inhibition of infection of rectal mucosa by VSV/HIV by directapplication of a 3 mM solution of the carbohydrate-lipid constructdesignated Gb₃-sp2-Ad-DOPE (III)(copy number HIV-1 cDNA)(n=4).

FIG. 14. Inhibition of infection of vaginal mucosa by VSV/HIV by directapplication of a 3 mM solution of the carbohydrate-lipid constructdesignated Gb₃-sp2-Ad-DOPE (III)(copy number HIV-1 cDNA)(n=4).

DETAILED DESCRIPTION

The specification accompanying international application no.PCT/NZ2005/000052 (publication no. WO 2005/090368) describes thepreparation and use of water soluble carbohydrate-lipid constructs. Inone example of the use of these constructs, qualitative and quantitativechanges in the surface antigen expression of red blood cells (RBCs) iseffected to provide quality control cells (e.g. SECURACELL™) for use invalidation of blood grouping.

Naturally occurring glycoconjugates, such as GSLs are not readilydispersible in water. Furthermore, it has been recognized that isolatedGSLs do not always retain the binding characteristics of themembrane-bound glycolipid. In fact it is stated in the specificationaccompanying international application no. PCT/CA97/00877 (publicationno. WO 98/23627) that solublised GSLs may have little or no bindingaffinity for compounds which bind strongly to the membrane bound GSL.

Mylvaganam and Lingwood (1999d) stated in the context of binding betweenthe GSL globotriaosyl ceramide (Gb₃) and the bacterial toxin verotoxinthat the reduction in binding affinity may be attributed toconformational changes influenced by the aglycone moiety. Whenincorporated in the plasma membrane conformational changes (favourableorientations) of the glycone moiety may be restricted by the plane ofthe membrane. The development of water soluble glycolipid mimics waspursued resulting in adamantyl conjugates which retained affinity forthe verotoxin receptor.

The carbohydrate-lipid constructs described in the specificationaccompanying international application no. PCT/NZ2005/000052 aredispersible in water and spontaneously incorporate into cell membranesas demonstrated by their use in the preparation of quality controlcells.

The present invention provides selected carbohydrate-lipid constructsthat are glycolipid mimics, but are dispersible in aqueous orbiocompatible media. The constructs may therefore be used in methods ofpreventing infection of cells by viruses in vivo.

The selected carbohydrate-lipid constructs are of the general formulaF-S₁-S₂-L where the alkylglycoside portion (F-S₁) is selected to providea ligand for a receptor expressed by a virus, and the spacer portion(S₁-S₂) is selected to provide a dispersible construct.

The constructs may function to inhibit both:

-   -   1. natural ligand-receptor binding (including “multivalent”        binding (Schengrund (2003)); and    -   2. post-binding events essential to infection of the target host        cell and subsequent replication of the virus.

As noted by Lund et al (2006) HIV targeting of CD4 and chemokineco-receptor expressing lymphoid and monocytic cells has long beenappreciated as the major mechanism of HIV-host cell interaction.

The gp120 receptor has also been shown to have an affinity in vitro fora number of GSLs including galactosyl ceramide, sulphogalactosylceramide and GM3 ganglioside (Feng et al (1996); Bhat et al (1993);Fantini et al (1998)). This binding affinity is characterized at leastin part by the nature of the carbohydrate moiety (glycotope) of the GSLligand. The receptor-GSL binding facilitates a post-CD4 binding event toallow the host cell entry of diverse HIV strains (Nehete et al (2002)).

Whilst not wishing to be bound by theory it is believed that inhibitingthe receptor-GSL binding event with a water soluble carbohydrate-lipidconstruct will inhibit host cell entry and viral infection of the cells.Furthermore, it is believed that inhibiting the post-CD4 binding eventin situ, i.e. at the co-receptor expressing surface of lymphoid andmonocytic cells will promote clearance of virus from an infectedsubject.

The in situ inhibition of the post-CD4 binding event may occur when thewater soluble carbohydrate-lipid construct is incorporated into the cellmembrane of the lymphoid and monocytic cells. The formation ofcarbohydrate-lipid construct enriched lipid microdomains on the hostcell surface may be central to both inhibiting viral infection andpromoting clearance of virus from an infected subject.

The methods of the invention may be effective against a plurality oftypes of HIV, including types X4 and R5). The ability of thecarbohydrate-lipid constructs to inhibit infection of cells by type R5HIV-1 is of particular significance as this is a strain of virus thatinitially infects susceptible subjects.

The carbohydrate-lipid constructs selected for use in the methods of theinvention are water soluble constructs that will partition, i.e.incorporate, into cell membranes. Furthermore, the preparation of thesesynthetic constructs excludes the use of substrates or reagents derivedfrom zoological sources. The carbohydrate-lipid constructs thereforeprovide advantages over semi-synthetic water soluble glycolipid mimicssuch as the adamantylGb₃ conjugates.

A number of receptor binding carbohydrate-lipid constructs may beeffective to inhibit infection or promote clearance of virus frominfected subjects. In addition to carbohydrate-lipid constructsincluding a Gb₃ carbohydrate moiety, constructs including the glycotopeof the GM3 carbohydrate moiety may also prove effective inhibitors ofHIV infection and promote clearance of the virus from an infectedsubject. Methods comprising the administration of two or more watersoluble carbohydrate-lipid constructs are contemplated.

The use of the carbohydrate-lipid constructs in the methods of theinvention is believed to be particularly advantageous because of theability of the constructs to incorporate non-specifically into themembranes of cells in vivo. The non-specific modification of cells invivo may permit multivalent binding and the adherence of the virus tocells in which the virus is unable to replicate.

Adherence to the cell surface of a cell via the carbohydrate portion ofthe carbohydrate-lipid construct may also result in the virus beingtrapped at the cell surface (cf. Asher et al (2005)). The ability of theimmune system to recognise and respond to the presence of virus maytherefore be augmented.

Although discussed with reference to the prevention and treatment ofsubjects with HIV infection, it will be recognised that a number ofviral infections are initiated by the adherence of the virus tocarbohydrates expressed at the surface of cells.

Schengrund (2003) and others have reviewed the development ofsaccharides as pharmacologic agents. As noted by this author, where itis determined that the expression of glycosphingolipids is necessary forinfection (Fantini et al (1993); Hanada (2005); Karlsson (1995); Isa etal (1997); Matrosovicha et al (1997); Miller-Podraza et al (2000);Suzuki (1994); Connor et al (1994); Matrosovich et al (1999); Willoughbyet al (1990)), the opportunity arises to interfere with the adherence ofthe virus (e.g. influenza virus, rotavirus) to the surface of targetcells.

EXPERIMENTAL

The carbohydrate-lipid constructs designated Gb₃-sp3-Ad-DOPE (I) andGb₃-sp2-Ad-DOPE (III) may be prepared and characterized in accordancewith the methods described mutatis mutandis in the specificationaccompanying international application number PCT/NZ2005/000052(publication no. WO 2005/090368) and summarized in Schemes I, II and IV.

The carbohydrate-lipid construct designated Gb₃-sp2-Ad-DOPE (III) mayalso be prepared by the method described below and summarized in SchemesIII and IV.

Scheme I:

(a) Cl₃CNN, DBU, CH₂Cl₂, −5° C., 64%; (b) Cl(CH₂)₃OH, BF₃*Et₂O, MS-4A,CH₂Cl₂, −5° C., 65%; (c) NaN₃, DSMO, 80° C., 20 h, 91%; (d) i)NaOMe/MeOH, 80%, ii) DMT, p-TsOH, DMF, 63%; (e) NaH, BnBr, 0° C., DMF,87%; (f) NaCNBH₃, HCl*Et₂O, MS-3A, −5° C., THF, 73%; (g) i) PPh₃, H₂O,THF, ii) MeOCOCF₃, Et₃N, THF, 84%

Scheme II:

(a) Cl₃CNN, K₂CO₃, CH₂Cl₂, 60%; (b) TMSOTf, MS-4A, CH₂Cl₂, 72% (c) H₂,10% Pd/C, MeOH; (d) Ac₂O/Py, 90% (e) MeONa/MeOH (f) NaOH/H₂O, 96%

Scheme III:

(a) Br₂, CH₂Cl₂, +4° C., 100%; (b) AgOTf, MS-4A, CH₂Cl₂, 78%; (c)MeONa/MeOH—CH₂Cl₂; (d) H₂, 10% Pd/C, MeOH, Boc₂O, 70%; (e) CF₃COOH(95%), 96%

Scheme IV:

(a), (b) DMF/CH₂Cl₂, Et₃N, 90-95%

Materials and Methods

TLC was performed on Silica gel 60 (Merck, Germany) precoated plates.Spots were visualized by treating with 5% aqueous orthophosphoric acidand subsequent heating to 150° C. in the case of carbohydrates or bysoaking in ninhydrin solution (3 g/l in 30:1 (v/v) butanol-acetic acid)in the case of amines.

Column chromatography was carried out on Silica gel 60 (0.040-0.063 mm,Merck, Germany). Gel chromatography was performed on Sephadex LH-20(Pharmacia, Sweden). Solvents were removed in vacuo at 30 to 40° C.

All solvents were from KhimMed (Russia). Molecular sieves (MS 3 Å and 4Å), trimethylsilyl trifluoromethanesulfonate, and triphenylphosphinewere from Aldrich (Germany). All hydrides,1,8-diazabicyclo[5,4,0]undec-7-ene (DBU), and trichloroacetonitrile werefrom Merck (Germany).

Anhydrous tetrahydrofuran (THF) and diethyl ether (Et₂O) were obtainedby distillation from lithium aluminium hydride (H₄AlLi). Dichloromethanefor glycoside synthesis was dried by distillation from phosphorouspentoxide and calcium hydride, and stored over molecular sieves MS 4 Å.Solid reagents were dried for 2 h in vacuo (0.1 mm Hg) at 20 to 40° C.

Deacetylation was performed according to Zemplen in anhydrous methanol.The solution of the acetylated compound was treated with 2 M sodiummethylate in methanol up to pH 9. When the reaction was completed, Na⁺ions were removed with cation exchange resin Dowex 50X-400 (H⁺) (Acros,Belgium). The solution was concentrated in vacuo.

Optical rotation was measured on a Jasco DIP-360 digital polarimeter at25° C.

Mass spectra were recorded on a Vision-2000 (Thermo Bioanalysis, UK)MALDI-TOF mass spectrometer using dihydroxybenzoic acid as a matrix.

¹H NMR spectra were recorded on a Bruker WM spectrometer (500 MHz) at25° C. Chemical shifts (δ, ppm) were recorded relative to D₂O (δ=4.750),CDCl₃ (δ=7.270), and CD₃OD (δ=3.500) as internal standards. The valuesof coupling constants (Hz) are provided. The signals in the ¹H NMRspectra were assigned by suppression of spin-spin interaction (doubleresonance) and 2D-1H,1H-COSY experiments.

Preparation of(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-acetyl-α-D-glucopyranosyltrichloroacetimidate (1)

Trichloroacetonitrile (12.1 ml, 121 mmol) and DBU (0.45 ml, 3 mmol) wereadded to a solution of 1a (7.68 g, 12.1 mmol) in dry dichloromethane(150 ml) at −5° C. The reaction mixture was stirred at −5° C. for 3.5 hand concentrated in vacuo.

Flash chromatography (2:1 to 1:2 (0.1% Et₃N) toluene-ethyl acetate) ofthe residue provided 1 (6.01 g, 63.9%) as a light yellow foam, R_(f)0.55 (2:1 toluene-acetone).

¹H NMR, CDCl₃: 1.95-2.2 (7s, 21H, 7Ac), 4.49 (d, 1H, J_(1,2)=8.07,H-1b), 4.91 (dd, 1H, J_(3,2)=10.3, J_(3,4)=2.8, H-3b), 5.05 (dd, 1H,J_(2,1)=3.5, J_(2,3)=9.3, H-2a), 5.12 (dd, 1H, J_(2,1)=8.07,J_(2,3)=10.3, H-2b), 5.32 (d, 1H, J_(4,3)=3, J_(4,5)<1, H-4b), 5.52 (t,1H, J_(3,2)=J_(3,4)=9.29, H-3a), 6.48 (d, 1H, J_(1,2)=3.5, H-1a), 8.64(s, 1H, HN═CCCl₃).

Preparation of3-chloropropyl-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-acetyl-β-D-glucopyranoside(2)

A mixture of 2.94 g (3.8 mmol) of trichloroacetimidate 1, 0.66 ml (7.5mmol) 3-chloropropanol, 50 ml dichloromethane, and 3 g of molecularsieves MS 4 Å was cooled to −5° C. An 8% solution of BF₃.Et₂O (0.4 mmol)in anhydrous dichloromethane was added drop wise with stirring.

After 30 min, the reaction mixture was filtered, diluted with chloroform(500 ml), and washed with water, saturated sodium hydrocarbonatesolution, and water to pH 7. The washed reaction mixture was dried byfiltration through a cotton layer and concentrated in vacuo.

Column chromatography on Silica gel (elution with 2.5:1 (v/v)toluene-ethyl acetate) resulted in 1.75 g (65%) of lactose derivative(2) as white foam. R_(f) 0.54 (2:1 toluene-acetone), R_(f) 0.50 (4:2:1hexane-chloroform-isopropanol), [α]_(D) −4° (c 1.0, CHCl₃), m/z 712.2(M⁺).

¹H NMR, CDCl₃: 1.95 (br. s, 5H, Ac, —CH₂—), 2.0-2.2 (6s, 18H, 6Ac), 3.52(m, 2H, —CH₂Cl), 3.63 (m, 1H, H-5a), 3.68 (m, 1H, OCHH—), 3.79 (t, 1H,J=9.3, H-4a), 3.88 (m, 1H, H-5b), 3.93-3.98 (m, 1H, OCHH—), 4.05-4.15(m, 3H, H-6a′, H-6b, H-6b′), 4.45 (d, 2H, H-1a, H-1b, J_(2,1)=7.83) 4.47(m, 1H, H-6a), 4.89 (dd, 1H, J_(2,3)=9.3, J_(2,1)=7.82, H-2a), 4.96 (dd,1H, J_(3,2)=10.5, J_(3,4)=3.42, H-3b), 5.11 (dd, 1H, J_(2,3)=10.5,J_(2,1)=7.83, H-2b), 5.21 (t, 1H, J=9.3, H-3a), 5.35 (dd, 1H,J_(4,3)=3.42, J_(4,5)<1).

Preparation of 3-azidopropyl(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-acetyl-β-D-glucopyranoside(3)

A mixture of 2.15 g (3 mmol) of trichloropropylglycoside 2, 0.59 g (9mmol) NaN₃, and 30 ml DMSO was maintained at 80° C. with stirring for 20h. The mixture was then diluted with chloroform (500 ml), washed withwater (4×100 ml), dried by filtration through a cotton layer, andconcentrated in vacuo.

Column chromatography on Silica gel (elution with 8:2:1hexane-chloroform-isopropanol) resulted in 1.96 g (91%) of glycoside (3)as a white foam, R_(f) 0.54 (2:1 (v/v) toluene-acetone), R_(f) 0.50(4:2:1 (v/v/v) hexane-chloroform-isopropanol), [α]_(D) −5.4° (c 1.0,CHCl₃), m/z 718.8 (M⁺).

¹H NMR, CDCl₃: 1.85 (m, 2H, —CH ₂—), 1.98-2.2 (7s, 21H, 7Ac), 3.36 (m,2H, —CH ₂N₃), 3.61 (m, 2H, H-5a, OCHH—CH₂—), 3.8 (t, 1H,J_(3,4)=J_(4,5)=9.29, H-4), 3.85-3.94 (m, 2H, OCHH—CH₂; H-5b), 4.05-4.17(m, 3H, H-6a, H-6a′, H-6b), 4.49 (d, 1H, J_(1,2=8.07), H-1a), 4.5 (m,1H, H-6b′), 4.51 (d, 1H, J_(1,2=8.07), H-1b), 4.9 (dd, 1H, J_(2,1)=8.07,J_(2,3)=9.29, H-2a), 4.97 (dd, 1H, J_(3,2)=10.27, J_(3,4)=3, H-3b), 5.12(dd, 1H, J_(2,1)=8.07, J_(2,3)=10.27, H-2b), 5.2 (t, 1H,J_(3,2)=J_(3,4)=9.29, H-3a), 5.36 (dd, 1H, J_(4,3)=3, J_(4,5)<1).

Preparation of 3-azidopropyl(4,6-O-benzylidene-β-D-galactopyranosyl)-(1→4)-β-D-glucopyranoside (4)

The lactoside 3 (1.74 g, 2.4 mmol) was deacetylated according to Zemplenand co-evaporated with toluene (2×30 ml). The residue was treated withα,α-dimethoxytoluene (0.65 ml, 3.6 mmol) and p-toluenesulfonic acid (50mg, to pH 3) in DMF (20 ml) for 3 h. The reaction mixture was thenquenched with pyridine, concentrated, and co-evaporated with o-xylene.

Column chromatography on Silica gel (elution with 9:1 (v/v)chloroform-isopropanol) and recrystalization (chloroform-methanol)resulted in 0.756 mg (62%) of benzylidene derivative (4). R_(f) 0.6 (5:1chloroform-isopropanol), [α]_(D)-25.7° (c 1.0, methanol), m/z 513.4(M⁺).

¹H NMR, CD₃OD: 2.06 (m, 2H, —CH ₂—), 3.45 (dd, 1H, J_(2,1)=J_(2,3)=9,H-2a), 3.61 (m, 1H, H-5a), 3.64 (m, 2H, —CH ₂N₃), 3.74-3.9 (m, 6H,OCHH—; H-3a, H-4; H-2b, H-3, H-5), 4.08-4.18 (m, 3H, H-6, H-6a′, OCHH—),4.34-4.44 (m, 3H, H-6b, H-6b′, H-4), 4.5 (d, 1H, J_(1,2)=7.9, H-1a),4.68 (d, 1H, J_(1,2)=8, H-1b), 5.82 (s, 1H, CHPh), 7.55-7.72 (m, 5H,CHPh).

Preparation of 3-azidopropyl(4,6-O-benzylidene-3-O-benzyl-β-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-D-glucopyranoside(5)

Sodium hydride in mineral oil (290 mg, 12 mmol) was slowly added in 4 to5 portions to a solution of 4 (726 mg, 1.5 mmol) in DMF (15 ml) at 0° C.with stirring. After 1 h, the ice bath was removed and benzyl bromidewas added drop wise. The mixture was stirred overnight. 10 ml ofmethanol was then added. After 1 h, the mixture was diluted withchloroform (500 ml), and washed with water (3×200 ml), dried byfiltration through a cotton layer, concentrated, and co-evaporated invacuo with o-xylene.

Column chromatography on Silica gel (elution with 10:1 toluene-ethylacetate) resulted in 1.24 g (87%) of lactose derivative 5 as white foam,R_(f) 0.56 (5:3 (v/v) hexane-ethyl acetate), [α]_(D)+10.8° (c 1.0,CHCl₃), m/z 963.8 (M⁺).

¹H NMR, CDCl₃: 1.85 (m, 2H, —CH ₂—), 2.91 (m, 1H, H-5), 3.33 (m, 1H,H-5a), 3.34-3.42 (m, 4H, H-2a, H-3, —CH ₂N₃), 3.55-3.62 (m, 2H, OCHH—;H-3a), 3.73 (dd, 1H, J_(2,1)=8, J_(2,3)=10, H-2b), 3.92-3.97 (m, 2H,H-4, OCHH—), 4.0 (br. d, 1H, J_(4,3)=3.6, H-4b), 4.34 (d, 1H,J_(1,2)=7.9, H-1a), 4.42 (d, 1H, J_(1,2)=8, H-1b), 5.43 (s, 1H, CH(Bd),7.14-7.50 (m, 30H, Ph).

Preparation of 3-azidopropyl(2,3,6-O-tri-O-benzyl-β-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-/3-D-glucopyranoside(6)

Hydrogen chloride in diethyl ether was added to a mixture of 5 (1.24 g,1.3 mmol), sodium cyanoborohydride (0.57 g, 9.1 mmol), and freshlyactivated molecular sieves MS 3 Å (33 g) in anhydrous THF (20 ml) untilthe evolution of gas ceased.

The mixture was stirred for 2 h, diluted with chloroform (300 ml),washed with water, saturated sodium hydrocarbonate solution, and waterto pH 7. The washed mixture was dried by filtration through a cottonlayer and concentrated in vacuo.

Column chromatography on Silica gel (elution with 20:1 to 7:3 (v/v)toluene-ethyl acetate) resulted in 0.91 g (65%) of lactose derivative 6as a white foam, R_(f) 0.42 (9:1 (v/v) toluene-acetone), [α]_(D)+17.8°(c 1.0, CHCl₃), m/z 965.8 (M⁺).

¹H NMR, CDCl₃: 1.85 (m, 2H, —CH ₂—), 2.39 (d, 1H, J=2.2, OH), 4.04 (br.s, 1H, H-4), 4.34 (d, 1H, J_(1,2)=7.9, H-1a), 4.42 (d, 1H, J_(1,2)=8,H-1b), 7.14-7.50 (m, 30H, Ph).

¹H NMR of acetylated analytical probe 6a, CDCl₃: 1.85 (m, 2H, —CH ₂—),4.34 (d, 1H, J_(1,2)=7.9, H-1a), 4.42 (d, 1H, J_(1,2)=8, H-1b), 5.5 (br.d, 1H, J_(4,3)=3.43, H-4), 7.14-7.50 (m, 30H, Ph).

Preparation of 3-trifluoroacetamidopropyl(2,3,6-O-tri-O-benzyl-β-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-D-glucopyranoside(7)

A mixture of derivative 6 (0.914 g, 0.94 mmol), triphenylphosphine (0.5g, 1.9 mmol) and THF (10 ml) was stirred for 0.5 h, 100 μl of wateradded, and the mixture stirred overnight. The reaction mixture was thenconcentrated and co-evaporated with methanol. The residue was dissolvedin methanol (15 ml) and triethylamine (30 μl) and methyltrifluoroacetate (0.48 ml, 4.7 mmol) added. The solution was held for 30min and then concentrated.

Column chromatography on Silica gel (elution with 5:1 to 1:1 (v/v)hexane-acetone) resulted in 0.87 g (84%) of lactose derivative 7 aswhite foam, R_(f) 0.49 (9:1 (v/v) hexane-acetone), [α]_(D)+17° (c 1.0,CHCl₃), m/z 1060.1 (M⁺+Na).

¹H NMR, CDCl₃: 1.88 (m, 2H, —CH ₂—), 2.40 (br. s, 1H, OH), 4.05 (br. s,1H, H-4), 4.36 (d, 1H, J_(1,2=7.8), H-1a), 4.40 (d, 1H, J_(1,2=7.6),H-1b), 7.10-7.35 (m, 30H, Ph).

Preparation of 2,3,4,6-tetra-O-benzyl-β-D-galactopyranosyltrichloroacetimidate (9)

A mixture of galactose derivative 8 (2 g, 3.65 mmol),trichloroacetonitrile (1.75 ml, 17.55 mmol), anhydrous potassiumcarbonate (2 g, 14.6 mmol), and dichloromethane (4 ml) was stirred for22 h at room temperature under argon. The mixture was then filteredthrough a Celite layer and concentrated in vacuo. Column chromatographyon Silica gel (elution with 4:1 (v/v) hexane-ethyl acetate (1% Et₃N)resulted in 1.5 g (60%) of 9 as white foam, R_(f) 0.47 (7:3 (v/v)hexane-ethyl acetate containing 1% Et₃N) and 0.46 g (0.8 mmol, 23%) ofthe starting derivative 8, R_(f) 0.27 (7:3 (v/v) hexane-ethyl acetatecontaining 1% Et₃N).

¹H NMR (CDCl₃): 3.60-3.70 (m, 3H, H-3, H-6, H-6′), 3.75 (t, 1H,J_(5,6)=6.30, H-5), 3.98 (d, 1H, J_(4,3)=2.19, H-4), 4.08 (dd, 1H,J_(2,3)=9.73, J_(2,1)=7.95, H-2), 4.42 and 4.47 (ABq, 2H, J=12.00, PhCH₂), 4.63 and 4.95 (ABq, 2H, J=11.51, PhCH ₂), 4.72 (s, 2H, PhCH ₂), 4.80and 4.90 (ABq, 2H, J=10.95, PhCH ₂), 5.74 (d, 1H, J_(1,2)=7.95, H-1),7.22-7.35 (m, 20H, ArH), 8.62 (s, 1H, NH).

Preparation of 3-trifluoroacetamidopropyl(2,3,4,6-tetra-O-benzyl-α-D-galactopyranosyl)-(1→4)-(2,3,6-tri-O-benzyl-β-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-D-glucopyranoside(10)

A mixture of lactose derivative 7 (158 mg, 0.153 mmol),trichlroroacetimidate 9 (120 mg, 0.175 mmol), molecular sieves MS 4 Å(0.5 g), and dichloromethane (5 ml) was stirred for 30 min at roomtemperature under argon. 0.1 ml of a 1% (v/v) solution of trimethylsilyltrifluoromethanesulfonate in dichloromethane was then added. After 2 h,another 50 mg (0.073 mmol) trichlroroacetimidate 9 and 30 μl of a 1%(v/v) solution of trimethylsilyl trifluoromethanesulfonate indichloromethane were added. The reaction mixture was stirred overnightat +4° C., quenched with triethylamine (5 μl), filtered, andconcentrated in vacuo.

Column chromatography on Silica gel (elution with 12:1 to 1:1 (v/v)toluene-ethyl acetate) resulted in 170 mg (72%) of trisaccharide 10;R_(f) 0.56 (4:1 (v/v) toluene-ethyl acetate); [α]_(D)+30.8° (c 1.0,CHCl₃).

¹H NMR, CDCl₃: 1.78-1.89 (m, 2H, —CH ₂—), 4.34 (d, 1H, J_(1,2)=7.8,H-1a), 4.43 (d, 1H, J_(1,2=7.4), H-1b), 5.06 (d, 1H, J_(1,2)=3.0, H-1c),7.14-7.48 (m, 50H, Ph).

Preparation of 3-trifluoroacetamidopropyl(2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl)-(1→4)-(2,3,6-tri-O-acetyl-β-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-acetyl-β-D-glucopyranoside(11)

The catalyst 10% Pd/C (10 mg) was added to a solution of the protectedoligosaccharide 10 (73 mg, 0.047 mmol) in methanol (7 ml), the mixturedegassed, and the flask filled with hydrogen. The reaction mixture wasstirred for 1 h, filtered off from the catalyst through a Celite layer,and concentrated in vacuo. The dry residue was dissolved in pyridine (2ml), acetic anhydride (1 ml) added, and the mixture held for 3 h. Thesolvents were then evaporated and residue co-evaporated with toluene(4×2 ml).

Column chromatography on Silica gel (elution with 2:1 hexane-acetone)resulted in 43.5 mg (90%) of trisaccharide 11 as a white foam, R_(f)0.52 (2:1 hexane-acetone), [α]_(D)+30.4° (c 1.0, CHCl₃).

¹H NMR, CDCl₃: 1.87 (2H, m, CH₂); 1.99, 2.05, 2.05, 2.06, 2.07, 2.07,2.09, 2.09, 2.12, and 2.14 (10×3H, 10 s, 10 Ac); 3.37 and 3.52 (2×1H, 2m, 2 CHN); 3.63 (1H, ddd, J_(4,5)=9.8, J_(5,6)=4.9, J_(5,6)=2.0, H-5a);3.72 (1H, m, OCH); 3.77 (1H, ddd≈br. T, J_(4,5)<1, J_(5,6)=6.8,J_(5,6)=6.1, H-5); 3.79 (1H, dd, J_(3,4)=9.3, J_(4,5)=9.8, H-4); 3.87(1H, m, OCH); 4.02 (1H, dd≈br. d, J_(3,4)=2.5, J_(4,5)<1, H-4); 4.09(1H, dd, J_(5,6)=4.9, J_(6,6)=12.0, H-6a); 4.12 (1H, dd, J_(5,6)=5.6,J_(6,6′)=10.8, H-6c); 4.14 (1H, dd, J_(5,6)=6.8, J_(6,6′)=11.0, H-6b);4.17 (1H, dd, J_(5,6′)=8.6, J_(6,6′)=10.8, H-6′c); 4.45 (1H, dd,J_(5,6′)=6.1, J_(6,6′)=11.0, H-6′b); 4.49 (1H, ddd br. T, J_(4,5)<1,J_(5,6)=5.6, J_(5,6′)=8.6, H-5c); 4.50 (1H, d, J_(1,2)=7.8, H-1a); 4.55(1H, d, J_(1,2)=7.8, H-1b); 4.59 (1H, dd, J_(5,6′)=2.0, J_(6,6′)=12.0,H-6′a); 4.76 (1H, dd, J_(2,3)=10.8, J_(3,4)=2.5, H-3b); 4.86 (1H, dd,J_(1,2)=8.1, J_(2,3)=9.5, H-2a); 4.10 (1H, d, J_(1,2)=3.4, H-1c); 5.12(1H, dd, J_(1,2)=7.8, J_(2,3)=10.8, H-2b); 5.19 (1H, dd, J_(1,2)=3.4,J_(2,3)=11.0, H-2c); 5.22 (1H, dd≈T, J_(2,3)=9.5, J_(3,4)=9.3, H-3a);5.40 (1H, dd, J_(2,3)=11.0, J_(3,4)=3.4, H-3c); 5.59 (1H, dd≈br. d,J_(3,4)=3.4, J_(4,5)<1, H-4c); 7.09 (1H, m, NHCOCF₃).

Preparation of 3-aminopropylα-D-galactopyranosyl-(1→4)-β-D-galactopyranosyl-(1→4)-β-D-glucopyranoside(Gb₃-sp3) (12)

Sodium methylate (30 μl of 2 M solution in methanol) was added to asolution of trisaccharide (11) (43 mg, 0.042 mmol) in anhydrous methanol(3 ml) and held for 2 h. The solution was then concentrated in vacuo,water (3 ml) added, and the mixture held for 3 h. The mixture was thenapplied to a column (10×50 mm) with Dowex 50X4-400 (H⁺ cation exchangeresin.

The target compound was eluted with 1 M aqueous ammonia and the eluantconcentrated in vacuo. Lyophilization from water provided trisaccharide12 (23 mg, quant.) as a colorless powder. R_(f) 0.3 (100:10:10:10:2(v/v/v/v/v) ethanol-n-butanol-pyridine-water-acetic acid), [α]_(D)+42°(c 1; water), m/z 584.9 (M⁺+Na).

¹H NMR, D₂O: 1.98-2.05 (m, 2H, —CH ₂—), 3.17 (m, 2H, —CH ₂NH₂),3.33-3.35 (m, 1H, H-2a), 4.36 (m, 1H, H-5c), 4.53 (d, 2H, J=7.8, H-1a,H-1b), 4.97 (d, 1H, J_(1,2)=3.67, H-1c).

Preparation of 2-azidoethyl(3,4-di-O-acetyl-2,6-di-O-benzyl-α-D-galactopyranosyl)-(1→4)-(2,3,6-tri-O-benzyl-β-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-D-glucopyranoside (13)

To the solution of ethyl3,4-di-O-acetyl-2,6-di-O-benzyl-1-thio-β-D-galactopyranoside (550 mg,1.11 mmol) in dichloromethane (10 ml) was added Br₂ (57 μl, 1.11 mmol).The mixture was held for 20 min at room temperature, then concentratedin vacuo at room temperature and co-evaporated with anhydrous benzene(3×30 ml). The crude3,4-di-O-acetyl-2,6-di-O-benzyl-α-D-galactopyranosylbromide (14) wasused for glycosylation without purification.

The mixture of lactose derivative 15 (Sun et al (2006)) (500 mg, 0.525mmol), 1,1,3,3-tetramethylurea (300 μl), molecular sieves MS 4 Å (1 g),and dichloromethane (25 ml) was stirred for 30 min at room temperature.Silver trifluoromethanesulfonate (285 mg, 1.11 mmol), molecular sievesMS 4 Å (0.5 g), and the freshly prepared galactopyranosylbromide (14) indichloromethane (15 ml) were then added. The reaction mixture wasstirred overnight, filtered, and concentrated in vacuo.

Column chromatography on Silica gel (elution with 3:1 to 1:1 (v/v)hexane-ethyl acetate) resulted in 570 mg (79%) of trisaccharide 13,R_(f) 0.25 (2:1 (v/v) hexane-ethyl acetate); [α]_(D) +32° (c 0.8, CHCl₃)

¹H NMR, CDCl₃: 1.88, 1.94 (2s, 2Ac), 3.00 (dd, 1H, J_(5,6)=4.9,J_(6′,6)=8.4, H-6a), 3.19 (dd, J_(1,2)=8.5, J_(2,3)=8.9, H-2a),3.30-3.36 (m, 2H, —CHHN₃, H-6′a), 3.38-3.47 (m, 4H, H-5a, H-5, H-2b,H-6b), 3.48-3.54 (m, 1H, —CHHN₃), 3.61 (dd, 1H, J_(2,3)=8.9,J_(3,4)=9.2, H-3a), 3.69-3.75 (m, 3H, H-6′b, H-6c, —OCHH—), 3.85 (dd,1H, J_(5,6)=4.6, J_(6,6′)=11.0, H-6c), 3.89 (dd, 1H, J_(1,2=3.4),J_(2,3)=10.8, H-2c), 3.95 (dd, 1H, J_(3,4)=9.2, J_(4,5)=9.5, H-4),4.0-4.1 (m, 4H, —OCHH—, H-4, CH₂Ph), 4.25, 4.29, 4.32, 4.39 (4 d, 4×1H,J_(AB)=12, 4 —CHPh), 4.43 (d, 1H, J_(1,2)=7.6, H-1), 4.48 (d, 1H,J_(1,2=7.6), H-1), 4.54-4.62 (m, 5H, 4 —CHPh, H-5c), 4.71-4.84 (m, 4H, 4—CHPh), 4.89, 4.91, and 5.09 (3 d, 3×1H, 3 4 —CHPh), 5.15 (d, 1H,J_(1,2)=3.0, H-1c), 5.39 (dd, 1H, J_(2,3)=10.8, J_(3,4)=3.4, H-3c), 5.56(dd, 1H, J_(3,4)=3.4, J_(4,5=)0.9, H-4c), 7.14-7.48 (m, 40H, Ph).

Preparation of 2-aminoethylα-D-galactopyranosyl-(1→4)-β-D-galactopyranosyl-(1→4)-β-D-glucopyranoside(Gb₃-sp2) (16)

Sodium methylate (100 μl of 2 M solution in methanol) was added to asuspension of trisaccharide (13) (500 mg, 0.363 mmol) in anhydrousmethanol (50 ml). The mixture was stirred overnight at room temperature,quenched with acetic acid, and concentrated in vacuo.

Column chromatography on Silica gel (elution with 2:1 to 1:1 (v/v)hexane-ethyl acetate) resulted in 470 mg of trisaccharide (17), R_(f)0.5 (1:1 (v/v) hexane-ethyl acetate), [α]_(D)+36° (c 0.5, CHCl₃).

To a solution of trisaccharide (17) and Boc₂O ((150 mg, 0.91 mmol) inanhydrous methanol (50 ml) was added the catalyst 10% Pd/C (500 mg). Themixture was degassed and the flask filled with hydrogen. The reactionmixture was stirred for 3 h, filtered off from the Pd/C, andconcentrated in vacuo.

Column chromatography on Silica gel (elution with 6:5:1 (v/v/v)chloroform-ethanol-water) resulted in 160 mg (68%) of trisaccharide 18R_(f) 0.3 (6:5:1 (v/v/v) dichloromethane-ethanol-water). ¹H NMR, D₂O:1.45 (s, 9H, (CH₃)₃COCO—), 4.53 (d, 1H, J_(1,2)=7.8, H-1b), 4.58 (d, 1H,J_(1,2)=7.4, H-1b), 4.98 (d, 1H, J_(1,2)=3.0, H-1c).

The trisaccharide 18 was then treated with 95% CF₃COOH (5 ml, 10 min).Upon completion, the mixture was concentrated in vacuo, co-evaporatedwith toluene, and applied to a column (10×100 mm) of Dowex 50X4-400 (H⁺)cation exchange resin. The target compound was eluted with 1 M aqueousammonia and the eluant was concentrated in vacuo. Lyophilization fromwater provided trisaccharide 16 (135, quant.) as a colorless powder.R_(f) 0.35 (100:10:10:10:2 (v/v/v/v/v)ethanol-n-butanol-pyridine-water-acetic acid), [α]_(D)+25° (c 0.2;water).

¹H NMR, D₂O:: 3.32 (m, 2H, —CH₂NH₂), 3.40-3.45 (m, 1H, H-2a), 3.63 (dd,1H, J_(1,2)=7.9, J_(2,3)=10.3, H-2b), 3.66-3.78 (m, 5H, H-5a, H-3a, H-4,H-6c, H-6′c), 3.8 (dd, 1H, J_(3,4)=3.1, J_(3,2)=10.3, H-3b), 3.84 (m,2H, J_(5,6)=4.4, J_(5,6′)=7.9, H-5), 3.88-3.92 (m, 3H, H-2c, H-6b,—OCHH—), 3.96 (dd, 1H, J_(3,4)=3.3, J_(3,2)=10.3, H-3c), 3.98-4.03 (m,2H, H-6a, H-6′b), 4.06 (dd, 1H, J_(5,6)=2.2, J_(6,6′)=12.3, H-6′a), 4.08(dd, 1H, J_(3,4)=3.3, J_(4,5=)0.9, H-4c), 4.09 (d, 1H, J_(3,4)=3.1,H-4), 4.17-4.21 (m, 1H, —OCHH—), 4.41 (m, 1H, H-5c), 4.56 (d, 1H, J=7.9,H-1b), 4.60 (d, 1H, J=8.1, H-1a), 5.00 (d, 1H, J_(1,2)=3.9, H-1c).

Preparation of activated1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine (Ad-DOPE) (19)

A solution of DOPE (40 μmol) in chloroform (1.5 ml) and triethylamine (7μl) were added to a solution of bis(N-hydroxysuccinimidyl) adipate (200μmol) in dry N,N-dimethylformamide (1.5 ml). The mixture was kept for 2h at room temperature, quenched with acetic acid, and partiallyconcentrated in vacuo.

Gel filtration on Sephadex LH-20 (1:1 (v/v) chloroform-methanolcontaining 0.2% acetic acid) of the residue yielded the activated lipid(37 mg, 95%) as a colorless syrup; R_(f) 0.5 (6:3:0.5 (v/v/v)chloroform-methanol-water).

¹H NMR (2:1 CDCl₃-CD₃OD): 5.5 [m, 4H, 2x (—CH═CH—)], 5.39 (m, 1H,—OCH₂-CHO—CH₂O—), 4.58 (dd, 1H, J=3.67, J=11.98, —CCOOHCH—CHO—CH₂O—),4.34 (dd, 1H, J=6.61, J=11.98, —CCOOHCH—CHO—CH₂O—), 4.26 (m, 2H,PO—CH₂-CH ₂—NH₂), 4.18 (m, 2H, —CH ₂—OP), 3.62 (m, 2H, PO—CH₂-CH ₂—NH₂),3.00 (s, 4H, ONSuc), 2.8 (m, 2H, —CH ₂—CO (Ad), 2.50 [m, 4H, 2x (—CH₂—CO)], 2.42 [m, 2H, —CH ₂—CO (Ad)], 2.17 [m, 8H, 2x (—CH ₂—CH═CH-CH₂—)], 1.93 (m, 4H, COCH₂CH ₂CH ₂CH₂CO), 1.78 [m, 4H, 2x (COCH₂CH ₂—)],1.43, 1.47 (2 br. s, 40H, 20 CH₂), 1.04 (m, 6H, 2 CH₃).

Preparation of Gb₃-sp3-Ad-DOPE (I) and Gb₃-sp2-Ad-DOPE (III)

To a solution of activated DOPE (19) (10.5 μmol) in dichloromethane (300μl) was added (12) or (16) (10 μmol) in DMF (0.5 ml) and thentriethylamine (3 μl). The mixture was kept for 2 h at room temperature.Gel filtration on Sephadex LH-20 (1:1 (v/v) chloroform-methanol) of themixture yielded (I) or (III) (90-95%).

Gb₃-sp3-Ad-DOPE (I) was determined to have a molecular weight (MW) of1415.7 and ¹H NMR (CDCl₃/CD₃OD, 2:1), δ: 5.5 (m, 4H, 2x (—CH═CH—),5.43-5.39 (m, 1H, —OCH₂-CHO—CH₂O—), 5.13 (d, 1H, J=3.6, H-1 Gal),4.61-4.58 (m, 2H; J=7.1, H-1 (Gal); J=3.7, J=12.1, —CCOOHCH—CHO—CH₂O—),4.46 (d, J=7.9, H-1 Gal), 2.53-2.48 (m, 4H, 2x (—CH ₂—CO), 2.42-2.37 (m,4H, COCH ₂CH₂CH ₂CH₂CO), 2.21-2.16 (m, 8H, 2x (—CH ₂—CH═CH—CH₂—),2.00-1.95 (m, 2H, O—CH₂CH ₂CH₂—NH), 1.78 (m, 8H, COCH₂CH ₂CH ₂CH₂CO and2x (COCH₂CH ₂—), 1.50, 1.47 (2 bs, 40H, 20 CH₂), 1.05 (m, 6H, 2 CH₃)(FIG. 1).

Gb₃-sp2-Ad-DOPE (III) was determined to have a molecular weight (MW) of1415.7 and ¹H NMR (CDCl₃/CD₃OD, 2:1), δ: 5.5 (m, 4H, 2x (—CH═CH—),5.43-5.39 (m, 1H, —OCH₂-CHO—CH₂O—), 5.13 (d, 1H, J=3.6, H-1 Gal),4.61-4.58 (m, 2H; J=7.1, H-1 (Gal); J=3.7, J=12.1, —CCOOHCH—CHO—CH₂O—),4.46 (d, J=7.9, H-1 Gal), 2.53-2.48 (m, 4H, 2x (—CH ₂—CO), 2.42-2.37 (m,4H, COCH ₂CH₂CH₂CH ₂CO), 2.21-2.16 (m, 8H, 2x (—CH ₂—CH═CH-CH ₂—), 1.78(m, 8H, COCH₂CH ₂CH ₂CH₂CO and 2x (COCH₂CH ₂—), 1.50, 1.47 (2 bs, 40H,20 CH₂), 1.05 (m, 6H, 2 CH₃).

In Vitro Studies

Inhibition of Infection of Jurkat Cells with a Pseudoenvelope-Typed HIV

The ability of Gb₃-sp3-Ad-DOPE (I) to inhibit infection by apseudoenvelope-typed HIV having an outer envelope derived from the mousevesticular stomatitis virus (VSV) and having an ENV-minus modified HIVgenome derived from the X4 HIV-1 NL4-3 virus was evaluated.

The methods described mutatis mutandis in the publication of Lund et al.(2006) were used to evaluate the ability of 250 μM Gb₃-sp3-Ad-DOPE (I)to inhibit infection of Jurkat cells by the pseudoenvelope-typed HIV.The carbohydrate-lipid construct was demonstrated to inhibit infectionby the VSV pseudoenvelope-typed virus (FIG. 2).

The ability of AZT to inhibit infection was used as a positive control.

Dose Response for Inhibition of Infection with a Pseudoenvelope-TypedHIV

HIV-1_(IIIB), an X4 type, T-cell-tropic HIV virus, was sourced from theNational Institutes of Health AIDS Research and Reference ReagentProgram. The virus was handled in a Level III biocontainment facility. Amultiplicity of infection (m.o.i) of 0.7 was used.

Gb₃-sp3-Ad-DOPE (I) in powdered form was dissolved in phosphate bufferedsaline to provide a stock solution of 6 mM. The stock solution wasdiluted to 2 mM to provide a working concentration.

HIV-1_(IIIB) was incubated with Gb₃-sp3-Ad-DOPE (I) at concentrations of50 at 1000 μM for 1 hour at 37° C. prior to incubation with Jurkatcells. Incubations were in a total volume of 100 μL.

A suspension of Jurkat cells at a density of 5×10⁵ per mL in 100 μLcomplete RPMI1640 medium was incubated with a solution of untreated ortreated (pre-incubated with Gb₃-sp3-Ad-DOPE (I) virus for 1 hour at 37°C.

Incubated cells were washed four times with phosphate buffered salinewithout MgCl₂/CaCl₂ and then cultured in 2 mL of complete RPMI1640medium for a total of 5 days. On days 0, 3, 4 and 5 500 μL aliquots ofculture supernatant were taken.

Aliquots of culture supernatant were stored at −80° C. A determinationof the level of HIV p24 core protein was conducted by ELISA (Coulter)for the supernatant of the Day 4 supernatant.

Results for quadruplicate experiments (r=4) are presented in Table 1 andFIG. 3.

Results for triplicate experiments (r=3) are presented in Table 2 andFIG. 4.

Dose Response for Inhibition of Infection of Peripheral BloodMononuclear Cells with an R5 Type Monocyte-Tropic HIV Virus

HIV-1_(Ba-L), an R5 type, monocyte-tropic HIV virus, was pre-incubatedwith Gb₃-sp3-Ad-DOPE (I) at the concentrations provided in FIG. 5 for 1hour. PHA/IL-2-activated human peripheral blood mononuclear cells(PBMCs) obtained from a healthy volunteer donor were then infected byincubation with the pre-treated virus for 1 hour (n=4).

p24 antigen expression, a measure of productive HIV infection, wasmonitored 12 days after infection. Gb₃-sp3-Ad-DOPE (I) was observed toinhibit infection by HIV-1_(Ba-L) at 400 μM (p<0.05) with a half-maximalinhibitory activity (IC₅₀) of circa 200 μm.

Dose Response for Inhibition of Infection of Jurkat C Cells with X4HIV-1 Virus

HIV-1_(IIIB) was pre-incubated with Gb₃-sp3-Ad-DOPE (I) at theconcentrations provided in FIG. 6 for 1 hour. Jurkat C cells were theninfected by incubation with the pre-treated virus for 1 hour (n=4).

p24 antigen expression, a measure of productive HIV infection, wasmonitored 5 days after infection. Gb₃-sp3-Ad-DOPE (I) was observed toinhibit infection by HIV-1_(IIIB) at 400 μM (p<0.001) with ahalf-maximal inhibitory activity (IC₅₀) of circa 200 μM.

In Vivo Studies

Mouse Model

A mouse model of HIV infection of the rectal and vaginal mucosa was usedfor in vivo evaluation of Gb₃-sp2-Ad-DOPE (III). A pseudoenvelope-typedreplication-deficient VSV-G/NL4-3luc HIV-1 virus (VSV/HIV) approved foruse in level 2 biocontainment was used to validate the mouse model.

Replication deficient, VSV-G enveloped HIV-1 luciferase containingrecombinant virions were prepared by co-transfection of 293T cells withplasmids containing the VSV-G envelope and the HIV-1 genome lacking envand with the luciferase gene inserted into the nef gene.

10 μg of plasmid containing the envelope and 15 μg of plasmid containingthe HIV genome were mixed and added drop wise to 2.5×10⁶ 293T cellsplated 24 hours previously. Plates were incubated for 72 hours at 37° C.

Viral supernatant was collected, centrifuged for 10 min, filteredthrough a 0.45 μm filter and ultracentrifuged in 8 mL aliquots over 400μL 20% glucose for 1 hour at 19,000 rpm. Pelleted virions wereresuspended in 800 μL TNE buffer and stored at −80° C. until furtheruse. Virion content was determined by p24 ELISA.

To determine infectivity of virus, 2×10⁵ Jurkat C cells were plated in a96 well tray in triplicate in 100 μL complete RPMI media lacking phenolred. 20 μL volumes of virus plus media up to a total volume of 200 μLper well was then added.

Cells were incubated for 48 hours, then lysed using Promega cell culturelysis solution. 100 μL of luciferase assay substrate was added to 20 μLof the lysed cells and luciferase activity was measured using aluminometer.

To determine if the virus infected a mouse epithelial cell line, 1×10⁶NIH3T3 cells were infected with 25 and 75 μL aliquots of virus andincubated in DMEM for 2 hours. DNA was isolated and subjected to PCR asdescribed below.

To determine if the virus infected mouse mucosal tissue, male and femaleCD1 mice were challenged rectally and vaginally with 25 μL of virusadministered via pipette to the rectum and vaginal cavities ofeuthanized mice for 2 hours. Rectal and vaginal tissue was then removed.DNA was isolated via the Qiagen DNEasy Isolation Kit for 50 mg oftissue.

Primers used for PCR amplification of HIV cDNA were forward: LTR5′-GGGACTGGAAGGGCTAATTC-3′ and reverse: L15′-AGGCAAGCTTTATTGAGGCTTAAGC-3′. Primers used for nested PCR were forward: L25′-CTGTGGATCTACCACACACA AGGCTAC-3′ and reverse: LTR U3 5′-CTCCCTGGAAAGTCCCCAGC-3′. Real time PCR was conducted using the RocheLightCycler 2.0 using the FastStart DNA Master Plus SYBR Green I Kit.

The pseudoenvelope-typed replication-deficient VSV-G/NL4-3luc HIV-1virus (VSV/HIV) was demonstrated to infect both Jurkat and NIH3T3 cellsby luciferase assay (FIGS. 7 and 8) and PCR (FIGS. 9 a and 9 b).

The pseudoenvelope-typed replication-deficient VSV-G/NL4-3luc HIV-1virus (VSV/HIV) was demonstrated to infect both rectal and vaginalmucosa (FIGS. 10 a and 10 b).

Inhibition of Rectal Infection by VSV/HIV by Gel Formulation ofGb₃-Sp3-Ad-DOPE (III)

A preliminary trial of a carbopol-based gel was performed. A gelcontaining 3 mM Gb₃-sp2-Ad-DOPE (III) was applied to rectal mucosa ofCD1 mice for 30 minutes before a one and one half hour challenge withVSV/HIV. Tissue was removed and quick frozen in liquid nitrogen. A gelcontaining only PBS was used as a control (n=4).

DNA was isolated from the tissue using the Qiagen tissue kit.Quantitative real time PCR (qPCR) using the Roche Lightcycler wasperformed to determine copy number of HIV-1 genomes using primers forthe LTR region of HIV-1 and compared to a quantified HIV-1 cDNA standardcurve.

Detection of HIV cDNA copies indicated successful viral entry andreverse transcription of the HIV genome (FIG. 11).

Inhibition of Vaginal Infection by VSV/HIV by Gel Formulation ofGb₃-Sp2-Ad-DOPE (III)

A preliminary trial of a carbopol-based gel was performed. A gelcontaining 3 mM Gb₃-sp2-Ad-DOPE (III) was applied to vaginal mucosa ofCD1 mice for 30 minutes before a one and one half hour challenge withVSV/HIV. Tissue was removed and quick frozen in liquid nitrogen. A gelcontaining only PBS was used as a control (n=4).

DNA was isolated from the tissue using the Qiagen tissue kit.Quantitative real time PCR (qPCR) using the Roche Lightcycler wasperformed to determine copy number of HIV-1 genomes using primers forthe LTR region of HIV-1 and compared to a quantified HIV-1 cDNA standardcurve.

Detection of HIV cDNA copies indicated successful viral entry andreverse transcription of the HIV genome (FIG. 12).

Inhibition of Rectal Infection by VSV/HIV Direct Application ofGb₃-Sp2-Ad-DOPE (III)

3 mM Gb₃-sp2-Ad-DOPE (III) was applied directly to rectal mucosa of CD1mice for 30 minutes before a one and one half hour challenge withVSV/HIV. Tissue was removed and quick frozen in liquid nitrogen (n=4).

DNA was isolated from the tissue using the Qiagen tissue kit.Quantitative real time PCR (qPCR) using the Roche Lightcycler wasperformed to determine copy number of HIV-1 genomes using primers forthe LTR region of HIV-1 and compared to a quantified HIV-1 cDNA standardcurve.

Detection of HIV cDNA copies indicated successful viral entry andreverse transcription of the HIV genome (FIG. 13).

Inhibition of Vaginal Infection by VSV/HIV Direct Application ofGb₃-sp2-Ad-DOPE (III)

3 mM Gb₃-sp2-Ad-DOPE (III) was applied directly to vaginal mucosa of CD1mice for 30 minutes before a one and one half hour challenge withVSV/HIV. Tissue was removed and quick frozen in liquid nitrogen (n=4).

DNA was isolated from the tissue using the Qiagen tissue kit.Quantitative real time PCR (qPCR) using the Roche Lightcycler wasperformed to determine copy number of HIV-1 genomes using primers forthe LTR region of HIV-1 and compared to a quantified HIV-1 cDNA standardcurve.

Detection of HIV cDNA copies indicated successful viral entry andreverse transcription of the HIV genome (FIG. 14).

Although the invention has been described by way of examples indicativeof its utility in the treatment of human subjects it should beappreciated that variations and modifications may be made to the claimedmethods with out departing from the scope of the invention. It will beunderstood that for a non-specific interaction, such as the interactionbetween the diacyl- or dialkyl-glycerolipid portion of thecarbohydrate-lipid constructs and a membrane, structural andstereo-isomers of naturally occurring lipids can be functionallyequivalent.

Where known equivalents exist to specific features, such equivalents areincorporated as if specifically referred to in this specification. Forexample, it is contemplated that diacylglycerol 2-phosphate could besubstituted for phosphatidate (diacylglycerol 3-phosphate) and that theabsolute configuration of phosphatidate could be either R or S.

TABLE 1 Inhibition of infection of Jurkat cells by pre-incubation of X4HIV-1 IIIB with carbohydrate- lipid construct designated Gb₃-sp3-Ad-DOPE(I) at the concentrations indicated (r = 4). control 50 μM 100 μM 200 μM400 μM 600 μM 800 μM 1000 μM 1 52.438 235.054 200.097 140.632 89.2021.008 1.008 1.209 2 229.629 220.79 224.205 14.267 2.213 1.008 0.2051.008 3 50.027 211.147 237.264 76.345 83.175 0.205 3.62 1.611 4 203.51347.415 28.531 221.393 2.816 0.807 128.578 0.205 mean 133.9018 178.6015172.5243 113.1593 44.3515 0.757 33.35275 1.00825 SD 96.05684 88.0073197.22216 88.70224 48.37245 0.380003 63.50024 0.591275 SEM 48.0284244.00366 48.61108 44.35112 24.18623 0.190001 31.75012 0.295638

TABLE 2 Inhibition of infection of Jurkat cells by pre-incubation of X4HIV-1 IIIB with carbohydrate- lipid construct designated Gb₃-sp3-Ad-DOPE(I) at the concentrations indicated (r = 3). control 50 μM 100 μM 200 μM400 μM 600 μM 800 μM 1000 μM 1 52.438 235.054 200.097 140.632 2.2131.008 1.008 1.209 2 229.629 220.79 224.205 76.345 83.175 1.008 0.2051.008 3 203.513 211.147 237.264 221.393 2.816 0.807 3.62 0.205 mean161.86 222.3303 220.522 146.1233 29.40133 0.941 1.611 0.807333 SD95.65768 12.0277 18.85523 72.67975 46.57034 0.116047 1.785571 0.531229SEM 55.22799 6.944197 10.88607 41.96168 26.8874 0.067 1.0309 0.306705

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The invention claimed is:
 1. A method of preparing an antiviralcomposition comprising the step of dispersing in water a construct ofthe formula F-S₁-S₂-L where: F is the glycoside1-O—(O-α-D-galactopyranosyl-(1→4)-O-β-D-galactopyranosyl-(1→4)-β-D-glucopyranosyl(Gb₃); S₁ is 2-aminoethyl, 3-aminopropyl, 4-aminobutyl or 5-aminopentyl;S₂ is —CO(CH₂)₃CO—, —CO(CH₂)₄CO— or —CO(CH₂)₅CO—; and L isphosphatidylethanolamine.
 2. The method of claim 1 where the antiviralcomposition is suitable for intravascular administration.
 3. The methodof claim 2 where L is1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine.
 4. The method ofclaim 3 where the construct has the structure:

where M is a monovalent cation.
 5. The method of claim 2 where theconstruct has the structure:

where M is a monovalent cation.
 6. The method of claim 3 where theconstruct has the structure:

where M is a monovalent cation.
 7. The method of claim 2 where theconstruct has the structure:

where M is a monovalent cation.